Abstract

The substrate for a solid state fermentation was composed of fresh cassava
roots as the carbohydrate source, urea to provide ammonia, diammonium
phosphate as a combined source of ammonia and phosphorus, and yeast (Saccharomyces cerevisiae) as the fermenting organism. The treatments were variants on the
traditional system used by farmers in the process of making rice “wine”;
prior steaming of the cassava root; no steaming; anaerobic system (no air access);
aerobic, spreading
the substrate in thin layers (about 10 cm to facilitate air exposure.

The levels of true protein increased from 2 to 7% in DM after 7 days of
incubation, representing a conversion of crude to true protein of
approximately 70%. There appeared to be no benefits from: (i) steaming the
cassava root prior to fermentation; and (ii) an aerobic system, providing
access to air during the fermentation as opposed to anaerobic conditions
with the substrates held in a sealed plastifor c bag. Oven–drying at 100°C
for 24h of samples of
cassava root containing urea, DAP and yeast, prior to or after fermentation,
led to varying losses of N which decreased with the increase in duration of
the fermentation.

Introduction

In most tropical countries there is an imbalance in availability of feeds
rich in protein compared with those composed predominantly of carbohydrates.
As a result, livestock production systems depend to a considerable extent on
imports of protein -rich supplements, especially soybean meal.

An approach that would provide a partial solution to this problem is through
protein-enrichment of feeds rich in carbohydrates by solid-state
fermentation with micro-organisms. Khempaka et al (2014) showed that aerobic
fermentation of steamed cassava pulp (crude protein content 2.0% in DM) with
Aspergillus oryzae and urea (3.5% in DM of the pulp) resulted in a
product with 8.1% true protein in DM, which could be incorporated at up to
16% of an intensive broiler diet without affecting growth performance and
feed conversion.
Improved growth rates in Moo Laat pigs were reported by Manivanh and
Prreston (2016) when protein-enriched cassava root replaced ensiled Taro (Colocacia
ensiformis) in a diet based on ensiled banana psuedo stem>

The aim of the research described in this paper was to study diffeent ways
of protein enrichment of cassava roots based on the procedure
traditionally used by farmers to produce rice wine.

Materials and methods

Experiment 1

The substrate for the solid state fermentation was composed of fresh cassava
root as the carbohydrate source, urea to provide ammonia, diammonium
phosphate as a combined source of ammonia and phosphorus, and yeast (Saccharomyces cerevisiae) as the fermenting organism. There
were four treatments that were variants on the traditional system used by farmers in the
process of making rice “wine”: Steaming prior to fermentation
(ST) or not steamed (NST); aair access during fermentation (AA) or no air
access (NAA).

The treatments were arranged as a 2*2 factorial with 4 replications.
For each treatment/replicate, the basal substrate was 1 kg of freshly ground
cassava root (CR).

For ST,
the cassava root was steamed for 30 minutes, allowed to cool for 15 minutes
and then mixed with urea (2% DM basis), diammonium phosphate (0.8% DM
basis) and yeast (2% DM basis). NST: The same as ST but not steamed.
AN: After mixing the substrates were spread on a plastic sheet at a depth of about
2cm to facilitate access to air. NAN: After mixing, the substrates were
put in a plastic bag which was closed to prevent air entry.

Measurements

On days 0, 3 and 7, samples were taken from each treatment/replicate
and dried in an oven at 100°C for 24h to determine the DM content. The dried
samples were then analyzed for crude and true protein (AOAC 1990). For estimation of true protein, 0.5 g of the “oven-dried”
sample was put in a 125ml Erlenmeyer flask with 50 ml of distilled water,
allowed to stand for 30 minutes, after which 10ml of 10% TCA (trichloracetic
acid ) were added and allowed to stand for a further 20-30 minutes. The
suspension was then filtered through Whatman #54 paper by gravity. The
filtrate was then discarded and the remaining filter paper and suspended
substrate were transferred to a kjeldahl flask for standard estimation of
total N.

Statistical analysis

The data were analyzed using the general linear option in the ANOVA program
of the Minitab (2000) software. Sources of variation were: steamed (yes or
no), air (yes or no), interaction steam*air and error.

Experiment 2

This experiment was done to demonstrate the losses in nitrogen from the
fermentation substrates in experiment 1 when they were subjected to 100°C oven drying prior
to analysis for CP and TP.

The procedure for preparation of substrates was similar to that used for the NAN
treatment in experiment 1.
Prior to fermentation, and 3 and 7 days after fermentation started, samples
were analysed for crude and true protein using: (a) the fresh
material; and (b) the dried material after heating at 100°C for 24h.

Results and discussion

Experiment 1

Data on the compostion of the substrates are in Table 1.

Table 1.
Composition of substrates

Urea

DAP#

Yeast

Cassava root

DM, %

100

100

90

29.0

Nitrogen, % in DM

46

18

7.76

0.40

Crude protein, % in DM

280

113

48.5

2.5

#Phosphorus 20%

Crude protein

After adding and mixing the urea, DAP and yeast with the cassava root, and
prior to fermentation, the crude protein values were only slightly
higher than in the
cassava root (2% in DM) (Figure 1). After 3 days the crude protein had
increased to 7.5% and by 7 days it was 16% in DM.

Figure 1.
Mean values for crude and true protein according to days fermented
(there were no differences among fermentation systems so the data were
averaged for all systems)

True protein

There were no benefits in true protein content from steaming the
cassava root prior to fermentation and no differences between the aerobic
and anaerobic treatments (Table 2)

Table 2.
Mean values for true protein (% in DM) of substrate before and after 3 and 7 days of fermentation

Days

Aeration

Steaming

Aerobic

Anaerobic

Yes

No

0

2.19

2.28

2.32

2.32

3

5.16

5.21

4.90

4.90

7

7.04

7.61

6.69

6.87

It is assumed that during the process of fermentation
increasing amounts of nitrogen (from the urea and DAP) would be converted to
yeast, but that the overall level of “crude” protein would not change.
However, it was observed that at time zero (prior to fermentation), the
crude protein was only 3% increasing to 7.5% after 3 days and
to 16% after 7 days,. These results indicated that nitrogen was being lost by drying the
samples (at 100°C for 24h) prior to kjeldahl analysis, but that
the degree of loss decreased with time of fermentation reaching"zero" loss
after 7 days. The proposed
explanation is that in the process of fermentation, the action of the yeast
led to increasing proportions of urea-N and DAP-N being converted to
precursors of protein (ie: amino acids and peptides) and to protein per se,
and that these compounds were not destroyed (evaporated?) by 100°C drying of the samples
prior to determination of kjeldahl-N. The final value of 16% crude protein
in DM aftet 7 days fermentation is higher than the theoretical content of
N*6.25 which is about 13% in DM. The difference could be accounted for
by the loss of carbohydrate substrate which provided the energy for the
growth of the yeast. Losses of the order of 20% of substrate DM during a
similar 10-day
fermentation were observed by Sengxayalath Phoutnapha (2016, personal communication).

The results from experiment 2 (Figure 2) in which
kjeldahl-N was determined on fresh samples, or after drying the samples at
100°C for 24h ,connfirmed that nitrogen is lost when a mixed substrate of
fresh cassava root, urea, DAP and yeast is oven-dried for 24h at 100°C. At time zero, after addition of urea, DAP and yeast, the mean
CP in the fermentation substrates that were not oven-dried was 8.5% in DM,
reflecting the N contained in the cassava root, urea, DAP and yeast. After
24h of fermentation the CP remained the same indicating no loss of N over
this period. By contrast, the CP in the DM of samples oven-dried at 100°C
prior to fermentation (3% in DM) reflected only the protein provided by the
cassava root (2%) and the yeast (1%). After 24h fermentation, the CP in
oven-dried samples had risen to 4% in DM, indicating a similar tendeny to
that observed in Experiment 1 (ie: reduced loss of nitrogen after
commencement of fermentation).

Nitrogenous compounds arising from yeast fermentation od urea and DAP

The unknown feature of the research concerns the nature of the non-protein
nitrogenous compounds remaining at the end of the fermentation. This is not
likely to be an issue in the feeding of ruminants, but could be of
consequence when the objective is to produce “protein-enriched” cassava root
for feeding to monogastric animals (pigs, chickens and fish) for which
excessive levels of elemental or ionized ammonia could be toxic. This requires: (i) more detailed
analysis to identify the nature of the non-protein nitrogenous compounds
present at the end of the fermentation and not converted to yeast; and (ii)
improving the efficiency of the fermentation to ensure that all the
nitrogenous compounds are converted to true protein.

Conclusions

Fermenting fresh cassava roots with yeast, urea and DAP, increased the
levels of true protein from 2 to 7% in DM after 7 days of incubation,
representing a conversion of crude to true protein of approximately 70%.

There appeared to be no benefits from: (i) steaming the cassava root
prior to fermentation; and (ii) an aerobic system, providing air access
during the fermentation as opposed to anaerobic conditions with the
substrates fermented in a sealed plastic bag.

Oven–drying at 100C for 24h of samples of cassava root containing urea,
DAP and yeast, prior to or after fermentation, led to major losses of N
which decreased with the increase in duration of the fermentation.

Acknowledgements

This research was done by the senior author as part of the requirements for
the MSc degree in Animal Production "Improving Livelihood and Food Security
of the people in Lower Mekong Basin through Climate Change Mitigation" in
Cantho University, Vietnam. The authors acknowledge financial support for this
research from the MEKARN II project financed by Sida.